S727 phosphorylation status of non-tyrosine-phosphorylated and tyrosine-phosphorylated Stat1. (A) Bac1.2F5 macrophages were treated with 5 ng/ml IFN- for 20 min. Western blots of anti-Stat1 immunoprecipitates subsequently were analyzed for the relative amount of tyrosine-phosphorylated Stat1 by staining with anti-Stat1 serum which reveals the slower migrating, tyrosine-phosphorylated form. The same precipitates were also stained with anti-pS727 serum to reveal the relative amount of S727-phosphorylated Stat1 in tyrosine-phosphorylated, slower migrating, and non-tyrosine-phosphorylated, faster migrating Stat1. The position and amount of tyrosine-phosphorylated Stat1 was determined similarly by staining the same blot with anti-phosphotyrosine monoclonal antibody. (B and C) The influence of H7 (B) or LPS (C) pre-treatment on the kinetics of Stat1 tyrosine phosphorylation by IFN-. Bac1.2F5 macrophages were pre-treated with LPS or H7 for 20 min and subsequently stimulated for the indicated periods with IFN-. Determination of Stat1 S727 phosphorylation was with anti-pS727 as described for Figure 1. Tyrosine phosphorylation was determined similarly using Py20 anti-phosphotyrosine monoclonal antibody (B) or antiserum to tyrosine-phosphorylated Stat1 (C). Additionally, DNA binding of Stat1 dimer in the EMSA as an indication of tyrosine phosphorylation is shown in (C).